转录组学分析Fe3O4纳米颗粒胁迫下盐单胞菌XH26与四氢嘧啶代谢相关的差异表达基因

Transcriptomics reveals differentially expressed genes related to ectoine metabolism in Halomonas campaniensisXH26 under Fe3O4 nanoparticle stress

摘要:

【目的】探究Fe₃O₄纳米颗粒(Fe₃O₄ nanoparticles,Fe₃O₄ NPs)与坎帕尼亚盐单胞菌(Halomonas campaniensis) XH26共培养后的差异表达基因(differentially expressed genes,DEGs)，明确Fe₃O₄ NPs促进XH26胞内四氢嘧啶积聚量增长的分子机制。【方法】设置空白组(C,0 g/L)、低浓度组(L,0.01 g/L)、中浓度组(M,0.10 g/L)和高浓度组(H,0.50 g/L)的Fe₃O₄ NPs与菌株XH26共培养。采用Illumina Hiseq 300PE进行转录组测序，探究菌株不同浓度组的DEGs，并采用RT-qPCR验证关键的DEGs。【结果】与空白C组相比，M组四氢嘧啶的积累量提高了55.67%(708.87 mg/L)，M组和H组的亚铁离子和抗氧化能力显著升高，而H组的羟基自由基含量高于M组。转录组学分析显示，M组中与菌株XH26胞内代谢相关的DEGs富集于精/脯氨酸代谢(13个)、氮代谢通路(11个)、硫代谢通路(10个)，主要功能与四氢嘧啶合成通路(11个)、电子传递途径(7个)及抗氧化酶系(5个)相关。RT-qPCR验证了四氢嘧啶合成代谢关键基因lysC、asd和基因簇ectABC，精氨酸(arginine,Arg)代谢通路基因astA/B/D/E以及尿素循环基因argE/H的表达趋势，与RNA-seq测序结果相一致。【结论】四氢嘧啶是细菌细胞和生物大分子重要的稳定保护剂。Fe₃O₄ NPs胁迫下菌株XH26胞内的活性氧(reactive oxygen species,ROS)增多，且影响胞内的氨基酸和氮代谢过程。菌株XH26通过提升抗氧化能力，增加胞内的四氢嘧啶积聚量，以应对Fe₃O₄ NPs的胁迫作用。

Abstract:

[Objective] To studyhydroxyl radical the differentially expressed genes (DEGs) in Halomonas campaniensis XH26 after co-culture with Fe₃O₄ nanoparticles (NPs), and clarify the molecular mechanism of Fe₃O₄ NPs in increasing the ectoine accumulation in strain XH26. [Methods] Strain XH26 was co-cultured with low-, medium-, and high-concentration (0.01, 0.10, and 0.50 g/L respectively in L, M, and H groups) Fe₃O₄ NPs, and the strain cultured without Fe₃O₄ NPs (0 g/L) was taken as the control group (C). Transcriptome sequencing was performed by Illumina HiSeq 300PE. The DEGs between different groups were mined, and key genes were screened for RT-qPCR verification. [Results] Compared with group C, group M showed an increase of 55.67% (708.87 mg/L) in ectoine accumulation, and groups M and H showed increased ferrous ions and antioxidant capacity. The hydroxyl radical content in group H was higher than that in group M. The transcriptomics analysis showed that the DEGs between groups M and C were enriched in arginine/proline metabolism (13), nitrogen metabolism (11), and sulfur metabolism (10) pathways. They were mainly related to the ectoine synthesis pathways (11), electron transport pathways (7), and antioxidant enzyme systems (5). RT-qPCR was employed to verify the expression of lysC, asd, and ectABC involved in ectoine synthesis, astA/B/D/E in arginine metabolic pathway, and argE/H in urea cycle, which showed the results consistent with the results of RNA-seq. [Conclusion] Ectoine is an important stable protective agent for bacterial cells and biomacromolecules. Strain XH26 exposed to the stress of Fe₃O₄ NPs showed increased intracellular reactive oxygen species and altered amino acid/nitrogen metabolism processes. Strain XH26 increased the accumulation of ectoine to cope with the stress of Fe₃O₄ NPs by improving the antioxidant capacity.